CN102507824A - Analysis method for modification sites of polyethylene glycol modified protein - Google Patents

Abstract

The invention belongs to the field of biomedicine, and relates to an analysis method for modification sites of polyethylene glycol modified protein, which at least includes: performing enzymolysis of proteomic tool enzymes; separating enzymolysis fragments, which include actual modification site residue with dominant content rather than possible but non-actual modification site residue in terms of sequence and are connected with a polyethylene glycol portion, from enzymolysis fragments, which include actual modification site residue with subordinate content rather than possible but non-actual modification site residue in terms of sequence and are connected with the polyethylene glycol portion, by means of high performance liquid chromatography; determining the proportion of modification sites with the dominant content by a peak area normalization method; determining the positions of the modification sites with the dominant content by means of N-end sequencing; and the like. By the aid of the analysis method, the distribution and the proportion of the modification sites in the polyethylene glycol modified protein can be determined more conveniently, rapidly and accurately at low cost.

Description

The decorating site analytical approach of polyethyleneglycol modified protein

Technical field

The structure analysis method that relates to polyethyleneglycol modified protein that the present invention is general; The special polyethyleneglycol modified site analytical approach that relates to polyethyleneglycol modified protein, the more special analytical approach that relates to dominant polyethyleneglycol modified site in the polyethyleneglycol modified protein.

Background technology

Protein is the important biomolecule bioactive molecule of structure and function of earning a bare living, and a kind of especially important pharmacologically active molecule can be used for the treatment of human various diseases.Although protein drug has been compared many characteristics and advantage with the traditional chemical medicine, its half life period weak point, poor stability, immunogenicity are strong etc., and a series of shortcomings have also limited its further applying in clinical practice.To these shortcomings; Produce and the chemical modification technology that has developed protein; That wherein the most successful is polyglycol (the Polyethylene Glycol of protein; PEG) modification technique; And developed a series of marketed drug and treated marketed drug; The polyethylene glycol modified interferon α 2a product of representational commodity like Switzerland Luo Shi (Roche) company " Pegasys " by name, the polyethylene glycol modified interferon α 2b product of the commodity " PegIntron " by name of U.S.'s Schering Plough (ScheringPlough) company (at present being merged by MSD Corp.), the polyethyleneglycol modified G-CSF product etc. of the commodity " Pegfilgrastim " by name of (Amgen) company is pacified into by the U.S..

Although the pharmaceutical grade protein product of the polyethyleneglycol modified more corresponding unmodified of pharmaceutical grade protein product has been obtained better therapeutic clinically; Obtained bigger success commercial; The trend that in clinical practice, replaces unmodified protein matter drug products is gradually arranged; But polyglycol is ubiquitous multidigit point property and site isomerism in the modifying protein process; The inhomogeneity that can cause modified outcome to form, and the different modifying degree has different physicochemical properties, pharmacologically active and security features with the modified outcome of decorating site, therefore can have influence on the quality control and the safety and effectiveness of such medicine.

In order to guarantee the safe and effective and quality controllability of polyethyleneglycol modified pharmaceutical grade protein; It more under possible situation the structure-activity relationship of further studying polyethyleneglycol modified pharmaceutical grade protein; Thereby prepare as the medicine of the further technology innovation of clinical use for from the polyethylene glycol modified product potpourri, isolating the modified outcome isomeride that structure is single, pharmacologically active is the highest, toxicity is minimum; Be necessary the structure of polyethyleneglycol modified protein is analyzed and proved conclusively, especially will analyze and control with each decorating site proportion the decorating site distribution of polyethyleneglycol modified protein.

The method that prior art is analyzed polyethyleneglycol modified protein modification site mainly is enzymolysis mass spectroscopy and enzymolysis LC-MS method, and concrete principle is following.

Enzymolysis mass spectrum ratio juris is to select a kind of proteomics toolenzyme commonly used or the combination of several kinds of toolenzymes; Like trypsase (Trypsin), chymotrypsin (Chymotrypsin), Glu-C, Asp-N etc.; Under suitable separately enzymatic hydrolysis condition to protein successively or carry out enzymolysis simultaneously so that possible different modifying site amino acid residue be distributed in different theoretical enzymatic fragments (as amino polyethyleneglycol modified dose of possible decorating site be lysine residues all in the protein; The amino polyethyleneglycol modified dose of amino acid residue of a modifying protein N end in theory of N end, but because specificity is not high, lysine residue that also might the non-N end of modifying protein; Polyethyleneglycol modified dose of possible decorating site of sulfydryl is cysteine residues all in the protein).Mass spectrometer is arrived in suitable the processings enzyme-added desampling in back in that enzymolysis reaction and the mass spectrometer that suits detect, carries out the mass spectrum molecular weight detection of enzymatic fragment like Q-TOF mass spectrometer or MALDI-TOF mass spectrometer.With the theoretical enzymatic fragment sequence of testing result and corresponding unmodified protein matter medicine and molecular weight distribution result contrast thereof (utilize the credit of range protein group to analyse software, perhaps go up the protein group credit and analyse the website, as Www.expasy.orgAll can analyze and obtain theoretical enzymatic fragment sequence and molecular weight distribution result thereof); Search the fragment that lacks in the testing result, the possible decorating site amino acid residue that has in this deletion fragment sequence is exactly the decorating site of the actual modifying protein medicine of polyglycol.The shortcoming of the method is high to the accuracy requirement of Mass Spectrometer Method, if having omission enzymatic fragment or enzymatic fragment detection molecules amount inaccurate among the Mass Spectrometer Method result, all can have a strong impact on result's judgement or the result can't be judged; In addition owing to the enzymatic fragment that connects polyalkylene glycol moiety lacks in the Mass Spectrometer Method result, so this method also can't be confirmed different modifying site product shared ratio in whole decorating site products.

Enzymolysis LC-MS ratio juris also is earlier with one or more suitable proteomics toolenzymes, selects suitable separately enzymatic hydrolysis condition successively or simultaneously protein is carried out enzymolysis.The LC-MS appearance is arrived in the suitable processing enzyme-added desampling in back in the enzymolysis reaction and the LC-MS detection that suits, after selecting suitable liquid chromatography separation condition to make each enzymatic fragment separated from one another, uses the molecular weight of each enzymatic fragment of mass detector detection that is connected with liquid chromatography.Since connect sensing range that the enzymatic fragment molecular weight of polyalkylene glycol moiety exceeds mass detector detect less than; So equally can be with the theoretical enzymatic fragment sequence and the molecular weight distribution result contrast thereof of testing result with corresponding unmodified protein matter; Search the deletion fragment that does not detect corresponding molecular weight, thereby confirm in esse decorating site.Contrast liquid chromatography peak area testing result then; To all representatives only contain same actual decorating site residue and do not comprise maybe but non-actual decorating site residue and the chromatographic peak that connects the enzymatic fragment of polyalkylene glycol moiety carry out the peak area normalization method handles; With modified outcome shared ratio in whole modified outcomes of confirming each same actual decorating site, also promptly confirm the ratio of each decorating site.Though the method customer service above-mentioned enzymolysis mass spectroscopy can only qualitative shortcoming that can not be quantitative; Mass detector omission enzymatic fragment or enzymatic fragment detection molecules amount are inaccurate to influence the shortcoming that qualitative results is analyzed but still exist, and also exists high performance liquid chromatography to separate the difficult shortcoming that the enzymatic fragment that respectively contains same actual decorating site and connect polyalkylene glycol moiety is separated fully.

Based on improvement to said method; Chinese patent CN200380103341.1 discloses the separation method of a kind of polyethylene glycol modified interferon α 2a (amino polyethyleneglycol modified dose of modified outcome) isomeride, also is the analytical approach of polyethylene glycol modified interferon α 2a decorating site.This method is initial gross separation polyethylene glycol modified interferon α 2a position isomer (being the decorating site isomeride) on the preparative liquid chromatography of weak cation exchange at first, then further separation point position isomeride on the preparative high performance liquid chromatography of strong cation exchange.Each isomeride of separating is only used respectively at lysine C terminal specific property enzymolysis, and do not carried out enzymolysis at the Trypsin of arginine C end enzymolysis, enzymolysis product carries out the MALDI-TOF Mass Spectrometer Method.Because connecting the lysine residue site of polyalkylene glycol moiety can not be by enzymolysis in enzymolysis process; So cause the theoretical enzymatic fragment that theoretical therewith enzymatic fragment is close on the theoretical enzymatic fragment that comprises this lysine residue site and the amino acid sequence; Link together and can not be become a big actual enzymatic fragment together with polyalkylene glycol moiety by the enzymolysis open form, thus cause the excessive MALDI-TOF of the exceeding Mass Spectrometer Method limit of whole big actual enzymatic fragment molecular weight and detect less than.Like this; As long as with theoretical enzymatic fragment sequence and molecular weight distribution contrast; In the MALDI-TOF testing result, search the theoretical enzymatic fragment of two disappearances that link together on the protein primary sequence; Just can confirm the lysine of one of them fragment C end, also be that a lysine before another fragment N terminal amino acid is actual decorating site.Behind the decorating site at the isomeride peak of confirming to separate on each high performance liquid chromatography, just can utilize the peak area normalization method to confirm each decorating site shared ratio in whole modified outcomes according to the peak area testing result of high performance liquid chromatography.Though this method has been made bigger improvement to preceding method; The result who obtains is more reliable, and (if multiple high performance liquid chromatography separating isomerism body is arranged, then each isomeride all need carry out the enzymolysis mass spectrophotometry but operation is comparatively loaded down with trivial details; Workload is bigger); Cost higher (lysine C terminal specific property enzymolysis Trypsin expensive, and since step many, analyze also big to polyethyleneglycol modified interferon-' alpha ' 2a requirement); Still high performance liquid chromatography is separated with Mass Spectrometer Method and have higher requirements; And can only solve the decorating site problem analysis of amido modified protein, can not solve the decorating site problem analysis of sulfydryl modification protein, so practical application have significant limitation.

Summary of the invention

The decorating site analytical approach that the purpose of this invention is to provide a kind of polyethyleneglycol modified protein;, high performance liquid chromatography separation enzymatic fragment incomplete influence result complete because of the theoretical enzymatic fragment detection of mass spectrum to exist in the technology that overcomes existing employing mass spectrum or LC-MS detection judge; And in most of the cases can not carry out quantitative shortcoming to each decorating site product proportion in whole modified outcomes, overcome the shortcoming that the testing process step is many, the time is long, cost is high simultaneously as far as possible.

For overcoming these shortcomings, the present invention provides the technical scheme that comprises the steps at least:

1) enzymolysis: primary enzymolysis at least, under enzymolysis fully and the condition that does not have non-specific enzymolysis, each possible decorating site is distributed in the different theoretical enzymatic fragments;

2) high performance liquid chromatography separates: at least high performance liquid chromatography separates behind each enzymolysis; Be distributed in the different chromatographic peaks with the relatively actual enzymatic fragment that does not connect polyalkylene glycol moiety of the enzymatic fragment that finally makes actual connection polyalkylene glycol moiety; And finally make at least include only dominant actual decorating site residue on the content on the sequence and do not comprise maybe but non-actual decorating site residue and the enzymatic fragment (hereinafter to be referred as the A fragment) that connects polyalkylene glycol moiety relatively include only actual decorating site residue less important on the same content on each sequence and do not comprise maybe but non-actual decorating site residue and connect the enzymatic fragment (hereinafter to be referred as the B fragment) of polyalkylene glycol moiety be distributed in the different chromatographic peaks;

3) confirm the decorating site ratio: to A fragment and B fragment 2) chromatographic resolution in final corresponding chromatographic resolution peak carry out peak area and add and handle; Not the peak area of the chromatographic peak that obtains of homogeneous chromatographic resolution need by add again after converting with the peak area of a chromatographic resolution with; And confirm that the maximum chromatographic resolution peak of peak area wherein accounts for that peak area adds and ratio, to confirm dominant actual decorating site proportion in whole decorating sites;

4) confirm the decorating site position: collect the A fragment the sample at final corresponding chromatographic resolution peak carry out the N terminal sequence and measure, compare with theoretical enzymolysis sequence and confirm the position of dominant decorating site in protein sequence.

In the enzymolysis of technique scheme, preferably possible decorating site is distributed in the different theoretical enzymatic fragments through primary enzymolysis.The selectable proteomics toolenzyme of enzymolysis process is not limited to but one or more combination among the preferred Trypsin, Chymotrypsin, Glu-C, Asp-N.But because Chymotrypsin enzymolysis site is too much, not exclusively with non-special, the price of Asp-N is high easily for enzymolysis process, so preferred toolenzyme is one or both the combination among Trypsin, the Glu-C.Again because the enzymolysis of Glu-C receives the influence of damping fluid kind, pH and time bigger, so most preferred toolenzyme is Trypsin.In enzymolysis process, preferably protein is handled, be more preferably at and with denaturant protein handled before the enzymolysis, so that enzymolysis process is more complete with denaturant urea.The ratio of the damping fluid of enzymolysis, pH, temperature, time, enzyme-to-substrate can reference tool enzyme manufacturer the product operation instruction; But ammonium bicarbonate enzymolysis damping fluid (pH8.0) for the preferred 2-20mmol/L of Trypsin; 37 ℃ hydrolysis temperature; 18-24 hour enzymolysis time, mass ratio 1: 10-1: the ratio of 40 enzyme-to-substrate.

In the high performance liquid chromatography of technique scheme separates, require to separate finally making between A fragment and each the B fragment separated from one anotherly at least through a step or multistep high performance liquid chromatography, it doesn't matter but whether separate between the B fragment of different actual decorating sites.Preferred RPLC of high performance liquid chromatography and molecular exclusion high performance liquid chromatography; Under the bigger situation of A fragment and each B fragment molecular weight difference, be more preferably molecular-exclusion chromatography; Because the influence factor that the molecular exclusion high performance liquid chromatography separates is less; It is easier that the research of separation condition obtains, and chromatogram is more directly perceived, is convenient to result's analysis and judgement.But because the enzymatic fragment molecular weight that contains polyalkylene glycol moiety in the enzymolysis potpourri is bigger; So should select wide-aperture efficient liquid phase chromatographic stuffing to separate, wherein the filler that can select of molecular exclusion high performance liquid chromatography includes but not limited to TSK-GEL G2000SW, TSK-GEL G2000SW _XL, TSK-GEL Super G2000, TSK-GEL G3000SW, TSK-GELG3000SW _XL, TSK-GEL Super G3000, Protein-Pak 125, Protein-Pak 200SW, Protein-Pak 300SW.

High performance liquid chromatography in technique scheme separates in the collection of illustrative plates that obtains, and the actual judgement that goes out the peak position that connects each enzymatic fragment of polyalkylene glycol moiety can be adopted one of following three kinds of methods:

1) retention time comparison method

After each molecular exclusion high performance liquid chromatography separates; At the pure article of protein of unmodified and the pure article of polyethyleneglycol modified protein of molecular weight homogeneous on the performance liquid chromatographic column respectively under same chromatographic fractionation system and the chromatographic separation condition; Confirm both retention times, the enzymatic fragment that then connects polyalkylene glycol moiety behind this polyethyleneglycol modified protein digestion should be at this between the two at same chromatographic fractionation system and the retention time under the chromatographic separation condition.

2) electrophoresis dying method

No matter be after each molecular exclusion high performance liquid chromatography separates; Still after each RPLC separates; Still after the high performance liquid chromatography of each other types separates; Each chromatographic peak to collecting carries out the SDS-PAGE electrophoresis detection respectively, successively carries out coomassie brilliant blue R250 dyeing and iodine staining behind the electrophoresis.Wherein coomassie brilliant blue R250 is to well known to a person skilled in the art method to the method that protein or polypeptide dye, and the method for iodine staining also can be with reference to the report of pertinent literature, or adopts following method:

Gel behind the electrophoresis is taken off; Immerse in the electrophoresis destainer and soaked 30 minutes; Take out then and put into about 10 minutes of 5% (w/v) barium chloride solution immersion, put into iodine staining liquid (saturated solution that solid iodine and 30% alcohol water blend are mixed with) dyeing 3-5 minute again.Gel after needing at last to dye is put into destainer and is soaked rinsing in 1-2 minute again and go just can take a picture and scanning analysis behind the loose colour on the gel.

The just representative that same band only all can develop the color two kinds of whens dyeing connects the enzymatic fragment of polyalkylene glycol moiety, otherwise the representative that develops the color during not at iodine staining if when coomassie brilliant blue R250 dyes, develop the color does not connect the enzymatic fragment of polyalkylene glycol moiety.If a certain swimming lane has the band that when two kinds of dyeing, all develops the color, then contain the enzymatic fragment that connects polyalkylene glycol moiety in the chromatographic peak of this swimming lane representative.

3) mass spectroscopy

No matter be after each molecular exclusion high performance liquid chromatography separates; Still after each RPLC separates; Still after the high performance liquid chromatography of each other types separates; To each chromatographic peak of collecting respectively desalination carry out the mass spectrum molecular weight detection after taking off organic solvent, record molecular weight greater than the peak of polyethyleneglycol modified agent molecule amount for connecting the enzymatic fragment peak of polyalkylene glycol moiety.

In the peak area of technique scheme adds and handles, if the A fragment separated fully in a chromatographic resolution with the B sheet is intersegmental, just then simply add with passable as long as will only contain the peak area of their chromatographic peak respectively.But if can not separate fully; Then need the peak area through the chromatographic peak that only contains A fragment or B fragment (whether it doesn't matter in the intersegmental separation of B sheet of different actual decorating sites) that just obtains after enzymolysis repeatedly and the chromatographic resolution is converted processing, it is the peak area in the same chromatographic resolution that all adding should be converted with peak area.Below the conversion disposal route is described for example, those skilled in the art after understanding following example, need not creative work just can be to other similar or more complicated situation convert processing.The peak area that should be pointed out that the enzymatic fragment that does not contain actual decorating site need not be considered when conversion.

Example 1:

1) enzymolysis high performance liquid chromatography separation case

The situation of the chromatographic peak of gained was as shown in table 1 below after each time enzymolysis high performance liquid chromatography separated, and the implication that code name is represented in the table is:

X1: which time enzymolysis and corresponding thereafter high performance liquid chromatography separate;

X2: the separating obtained chromatographic peak b of a time enzymolysis high performance liquid chromatography that this enzymolysis high performance liquid chromatography separates is expressed as a, b;

Y1: the actual decorating site that contains in the enzymatic fragment that this peak contains;

Y2: contain in the enzymatic fragment that this peak contains maybe but non-actual decorating site;

Z1: whether each enzymatic fragment that this peak is corresponding respectively is connected with a peg molecule, and (enzymatic fragment can not be connected with two above peg molecules; Each enzymatic fragment or all be connected with a peg molecule, or do not connect peg molecule);

Z2: whether only contain an enzymatic fragment in this peak.

Each time of table 1 enzymolysis high performance liquid chromatography separates the situation of the chromatographic peak of back gained

2) decorating site ratio result of calculation: the peak area of chromatographic peak of enzymolysis chromatographic resolution gained is converted by the first time

The shared ratio of a decorating site is: A11/ (A11+A12)

The shared ratio of b decorating site is: A12/ (A11+A12)

Wherein Aab representes the peak area of the peak b of gained after a time enzymolysis chromatographic resolution.

Example 2:

1) enzymolysis high performance liquid chromatography separation case

The situation of the chromatographic peak of gained was as shown in table 2 below after each time enzymolysis high performance liquid chromatography separated, and the code name implication is like example 1 in the table.

Each time of table 2 enzymolysis high performance liquid chromatography separates the situation of the chromatographic peak of back gained

2) decorating site ratio result of calculation: the peak area of chromatographic peak of enzymolysis chromatographic resolution gained is converted by the first time

The shared ratio of a decorating site is: A11/ (A11+A12)

The shared ratio of b decorating site is: A12 * A21/ (A21+A22)/(A11+A12)

The shared ratio of c decorating site is: A12 * A22/ (A21+A22)/(A11+A12)

Wherein Aab representes the peak area of the peak b of gained after a time enzymolysis chromatographic resolution.

When A11/ (A11+A12)＞0.5; Can confirm directly that a is the actual decorating site that has comparative advantage, and can not consider the ratio of other actual decorating sites and confirm that the actual decorating site a shared ratio in whole actual decorating sites that has comparative advantage is A11/ (A11+A12).

Example 3:

1) enzymolysis high performance liquid chromatography separation case

The situation of the chromatographic peak of gained was as shown in table 3 below after each time enzymolysis high performance liquid chromatography separated, and the code name implication is like example 1 in the table.

Each time of table 3 enzymolysis high performance liquid chromatography separates the situation of the chromatographic peak of back gained

2) decorating site ratio result of calculation: the peak area of chromatographic peak of enzymolysis chromatographic resolution gained is converted by the first time

The shared ratio of a decorating site is: A11/ (A11+A12+A13)

The shared ratio of b decorating site is: A12 * A21/ (A21+A22)/(A11+A12+A13)

The shared ratio of c decorating site is: A12 * A22/ (A21+A22)/(A11+A12+A13)

The shared ratio of e decorating site is: A13 * A31/ (A31+A32)/(A11+A12+A13)

The shared ratio of f decorating site is: A13 * A32/ (A31+A32)/(A11+A12+A13)

Wherein Aab representes the peak area of the peak b of gained after a time enzymolysis chromatographic resolution.

When A11/ (A11+A12+A13)＞0.5; Can confirm directly that a is the actual decorating site that has comparative advantage, and can not consider the ratio of other actual decorating sites and confirm that the actual decorating site a shared ratio in whole actual decorating sites that has comparative advantage is A11/ (A11+A12+A13).

Example 4:

1) enzymolysis high performance liquid chromatography separation case

The situation of the chromatographic peak of gained was as shown in table 4 below after each time enzymolysis high performance liquid chromatography separated, and the code name implication is like example 1 in the table.

Each time of table 3 enzymolysis high performance liquid chromatography separates the situation of the chromatographic peak of back gained

2) decorating site ratio result of calculation: the peak area of chromatographic peak of enzymolysis chromatographic resolution gained is converted by the first time

The shared ratio of a decorating site is: A11 * A21/ (A21+A22) * A31/ (A31+A32)/(A11+A12+A13)

The shared ratio of b decorating site is: A11 * A21/ (A21+A22) * A32/ (A31+A32)/(A11+A12+A13)

The shared ratio of c decorating site is: A11 * A22/ (A21+A22)/(A11+A12+A13)

The shared ratio of e decorating site is: A12 * A41/ (A41+A42)/(A11+A12+A13)

The shared ratio of f decorating site is: A12 * A42/ (A41+A42) * A51/ (A51+A52)/(A11+A12+A13)

The shared ratio of g decorating site is: A12 * A42/ (A41+A42) * A52/ (A51+A52)/(A11+A12+A13)

The shared ratio of k decorating site is: A13 * A71/ (A71+A72)/(A11+A12+A13)

The shared ratio of l decorating site is: A13 * A72/ (A71+A72)/(A11+A12+A13)

Wherein Aab representes the peak area of the peak b of gained after a time enzymolysis chromatographic resolution.

Technique scheme the N terminal sequence measure; Preferred 3-8 the amino acid of N end of measuring; Be more preferably and measure 4-6 amino acid of N end; Because the amino acid number of measuring is unfavorable for the judgement of enzymatic fragment complete sequence less, and the amino acid number of measuring at most not only minute prolong, and the amino acid whose accuracy of subsequent measurements also can not get assurance with confidence level.

As to the improved optimal technical scheme of technique scheme, following steps are further required:

1) high performance liquid chromatography separates: at least high performance liquid chromatography separates behind each enzymolysis, is distributed in the different chromatographic peaks finally to make each B fragment;

2) confirm the decorating site ratio: the peak area of the chromatographic peak at each B fragment place is added and handles; The peak area that the peak area that calculates the chromatographic peak at each B fragment place accounts for the chromatographic peak at whole B fragments place add and ratio, to confirm each actual decorating site proportion in whole decorating sites;

3) confirm the decorating site position: the sample of collecting the chromatographic resolution peak at each B fragment place carries out the N terminal sequence to be measured, and compares with theoretical enzymolysis sequence and confirms each actual decorating site position in protein sequence.

In above-mentioned optimized technical scheme, when high performance liquid chromatography separates, not only require to separate finally making between A fragment and each the B fragment separated from one anotherly through a step or multistep high performance liquid chromatography, and require between the B fragment separated from one another.Therefore, higher to the requirement of high performance liquid chromatography separation.

In the peak area of above-mentioned optimal technical scheme added and handles, if between A fragment and each the B fragment, and each B fragment separated in a chromatographic resolution each other fully, just then as long as the peak area of their place chromatographic peaks is simply added with passable.But if can not separate fully, then need to through enzymolysis repeatedly with separate after the A fragment that just obtains or the peak area of each B fragment convert processing, concrete grammar such as preceding said for example.

Adopt above-mentioned basic technology scheme and optimal technical scheme; Because comparing on molecular weight and hydrophobicity with the enzymatic fragment that does not comprise actual decorating site residue and be connected polyalkylene glycol moiety, A fragment and each B fragment have than big-difference; Quantitatively obviously still less, so select suitable high performance liquid chromatography separation condition one to make between A fragment and each the B fragment separated from one another surely; And separate step number or optimize the high performance liquid chromatogram separation condition through further increasing high performance liquid chromatogram, also can further make between each B fragment separated from one anotherly, be distributed in the different chromatographic peaks.Because the order-checking of N end is different from Mass Spectrometer Method, needs only and remove organic solvent, guarantee applied sample amount that the protein in the last appearance or its fragments sequence necessarily can be measured and obtain again.Therefore, in case separated from one another between A fragment and the B fragment, collect and only to contain the chromatographic peak of A fragment and after proper process, to carry out the order-checking of N end again, with regard to one confirm dominant decorating site surely position and ratio; In case and further separation each other between the B fragment is collected the chromatographic peak that only contains each B fragment and after suitably handling, carried out N again and holds order-checking, just can further confirm the position and the ratio of other decorating sites.So the present invention can confirm the distribution and the ratio of decorating site in the polyethyleneglycol modified protein convenient, quick, accurately, cheaply.

The term that uses among the present invention " protein sequence " or " fragment sequence " particularly point out primary structure or the primary sequence that is meant protein or its enzymatic fragment like nothing, also are each amino acid whose putting in order of constitutive protein matter or its enzymatic fragment.

The term that uses among the present invention " polyethyleneglycol modified dose "; Also be " PEG dressing agent "; Be meant the mono methoxy polyethylene glycol dressing agent; Also promptly with a hydroxyl of methoxyl sealing peg molecule one end, and with the activated polyethylene glycol molecule of gained behind the hydroxyl of the activation method activation other end that suits.Because the reactivity of hydroxyl self is very low, so the reactivity of the peg molecule behind overactivation is greatly improved, just can be called as " polyethyleneglycol modified dose ".About the selection of activated group, the mechanism of activation and polyethyleneglycol modified dose modification reaction mechanism of activation gained a lot of reports are arranged in the prior art, like U.S. Nektar company (former Shearwater company) apply for and obtained the authorization many pieces relevant polyethyleneglycol modified dose patent.

The term that uses among the present invention " polyethyleneglycol modified protein " (pegylated protein; PEG modified protein); Be also referred to as " polyethylene glycol conjugation protein " (PEG coupled protein; PEG conjugated protein), " pegylated protein ", " polyglycol long-acting protein ", be meant polyethyleneglycol modified agent molecule with the protein molecule chemical coupling to the product that forms.Polyethyleneglycol modified dose the group of participating in coupling reaction is its activated group of in reactivation process, introducing; The group of protein mainly is wherein free amino group, mercapto groups, carboxylic group or oh group, preferably amino group or mercapto groups.

The term that uses among the present invention " modified by polyethyleneglycol body " or " modified by polyethyleneglycol protein " are meant that a protein molecule only connects the polyethyleneglycol modified protein that a peg molecule forms; Corresponding " polyglycol is modified body more " or " the many modifying proteins of polyglycol " is meant that a protein molecule connects the polyethyleneglycol modified protein that a plurality of peg molecules form.The present invention carry out the decorating site dissecting needle right all be " modified by polyethyleneglycol body " or " modified by polyethyleneglycol protein ".If analyze " polyglycol is modified body " or " the many modifying proteins of polyglycol " arranged in the polyethyleneglycol modified protein more; Need to separate in advance to remove, remove based on principle mainly be mono-modified body with the bodies of modifying more in difference on the molecular weight or the difference on the hydrophilic and hydrophobic.

The term that uses among the present invention " decorating site " is meant the polyethyleneglycol modified dose of amino acid residue site in the protein sequence that is connected.

The term that uses among the present invention " possible decorating site " be meant in the protein sequence might be but not necessarily actual by the polyethyleneglycol modified dose of amino acid residue site of modifying or connecting, this is mainly determined by the type of the modification reaction kind with dressing agent.Specifically; Possible decorating site is lysine (Lys) residue all in the protein sequence during amino polyethyleneglycol modified dose of (like mPEG-SPA, mPEG-NHS, mPEG-ALD, mPEG-SS, mPEG-SC) modifying protein, and possible decorating site is halfcystine (Cys) residue all in the protein sequence during polyethyleneglycol modified dose of (like mPEG-MAL, mPEG-VS, mPEG-IA, mPEG-OPSS) modifying protein of sulfydryl.

The term that uses among the present invention " actual decorating site " is meant in the protein sequence actual in the polyethyleneglycol modified dose of amino acid residue site of modifying or connecting; It is included in the possible decorating site scope; Also,, quantity counts but being less than or equal to possible modifier bit by the type of modification reaction and the kind decision of dressing agent.

The term that uses among the present invention " dominant actual decorating site " is meant that polyethyleneglycol modified protein molecule shared molecule percentage in whole polyethyleneglycol modified protein molecules of the single decorating site of actual a certain modification is the decorating site of maximum ratio.

The term that uses among the present invention " the actual decorating site that has comparative advantage " is meant that polyethyleneglycol modified protein molecule shared molecule percentage in whole polyethyleneglycol modified protein molecules of a certain decorating site of actual single modification surpasses 50% decorating site.

The term that uses among the present invention " less important actual decorating site " is meant the polyethyleneglycol modified protein molecule decorating site that shared molecule percentage is not maximum ratio in whole polyethyleneglycol modified protein molecules of a certain decorating site of actual single modification.

The term that uses among the present invention " different chromatographic peaks " both had been included in the different chromatographic peak of retention time in the high performance liquid chromatography separation, also comprised the chromatographic peak during the homogeneous high performance liquid chromatography separates.

The term that uses among the present invention " final corresponding chromatographic resolution peak " is meant if the different chromatographic peak during repeatedly high performance liquid chromatography separates all comprises the enzymatic fragment that contains same actual decorating site; Possibly comprise the enzymatic fragment that contains other actual decorating site in addition, the corresponding chromatographic peak that only contains the enzymatic fragment of this same actual decorating site in the last chromatographic resolution of then selecting this moment.

Description of drawings

Fig. 1 is TSK-GEL G3000SW on the pure article of IIFNm _XL(the chromatogram that obtains behind the molecular exclusion performance liquid chromatographic column of 7.8mm * 30cm).

Fig. 2 is TSK-GEL G3000SW on the pure article of PEG-IIFNm1 _XL(the chromatogram that obtains behind the molecular exclusion performance liquid chromatographic column of 7.8mm * 30cm).

Fig. 3 is that Trypsin enzymolysis PEG-IIFNm1 product is through TSK-GEL G3000SW _XL(the chromatogram that the molecular exclusion performance liquid chromatographic column of 7.8mm * 30cm) obtains after separating; Retention time is the enzymatic fragment peak T1 of the peak of 7.142min for the connection polyalkylene glycol moiety among the figure, and retention time is the enzymatic fragment peak T2 of the peak of 7.561min for the connection polyalkylene glycol moiety.

Fig. 4 be connect polyalkylene glycol moiety the PEG-IIFNm185-121 fragment behind the Chymotrypsin enzymolysis again through TSK-GEL G3000SW _XL(7.8mm * 30cm) the molecular exclusion performance liquid chromatographic column separates the chromatogram that obtains, and retention time is the enzymatic fragment peak C1 of the peak of 7.597min for the connection polyalkylene glycol moiety among the figure.

Fig. 5 is that Trypsin enzymolysis PEG-IIFNm1 product is through Waters Symmetry 300C ₁₈(the chromatogram that 4.6 * 150mm) RPLC posts obtain after separating; Retention time is the enzymatic fragment peak T1 of the peak of 33.250min for the connection polyalkylene glycol moiety among the figure; Retention time is the enzymatic fragment peak T2 of the peak of 42.433min for the connection polyalkylene glycol moiety; Retention time is the enzymatic fragment peak T3 of the peak of 60.458min for the connection polyalkylene glycol moiety, and retention time is the enzymatic fragment peak T4 of the peak of 64.098min for the connection polyalkylene glycol moiety.

Fig. 6 be connect polyalkylene glycol moiety the PEG-IIFNm185-121 fragment behind the Chymotrypsin enzymolysis again through Waters Symmetry 300C ₁₈(4.6 * 150mm) RPLC posts separate the chromatogram that obtains, and retention time is the enzymatic fragment peak C1 of the peak of 10.825min for the connection polyalkylene glycol moiety among the figure.

Fig. 7 is the (chromatogram that obtains behind the molecular exclusion performance liquid chromatographic column of 8.0mm * 30cm) of Protein-Pak 200SW on the pure article of G-CSF.

Fig. 8 is the (chromatogram that obtains behind the molecular exclusion performance liquid chromatographic column of 8.0mm * 30cm) of Protein-Pak 200SW on the pure article of PEG-G-CSF.

Fig. 9 is a Trypsin enzymolysis PEG-G-CSF product through the Protein-Pak 200SW (chromatogram that the molecular exclusion performance liquid chromatographic column of 8.0mm * 30cm) obtains after separating; Retention time is the enzymatic fragment peak T1 of the peak of 7.058min for the connection polyalkylene glycol moiety among the figure, and retention time is the enzymatic fragment peak T2 of the peak of 7.560min for the connection polyalkylene glycol moiety.

Figure 10 is that Trypsin enzymolysis PEG-VEGF product is through Waters Symmetry 300C ₁₈(the chromatogram that 4.6 * 150mm) RPLC posts obtain after separating; Retention time is the enzymatic fragment peak T1 of the peak of 19.845min for the connection polyalkylene glycol moiety among the figure; Retention time is the enzymatic fragment peak T2 of the peak of 29.373min for the connection polyalkylene glycol moiety; Retention time is the enzymatic fragment peak T3 of the peak of 43.052min for the connection polyalkylene glycol moiety; Retention time is the enzymatic fragment peak T4 of the peak of 58.278min for the connection polyalkylene glycol moiety, and retention time is the enzymatic fragment peak T5 of the peak of 78.452min for the connection polyalkylene glycol moiety.

Embodiment

Through following embodiment enforcement of the present invention is described further, but embodiment of the present invention is not limited to following embodiment.

Embodiment 1: Interferon Alfacon-1 86 site cysteine mutant modified by polyethyleneglycol product decorating sites are analyzed (method one)

1, the acquisition of IIFNm and PEG-IIFNm1

(integrated interferon site 86 mutant, amino acid sequence IIFNm) is shown in SEQ ID No.1 for Interferon Alfacon-1 86 site cysteine mutants.Successively obtaining IIFNm with embodiment 1A, embodiment 2A, the disclosed raw material of embodiment 3A and method among the PCT/CN2007/003711 (also is disclosed MIFN among the PCT/CN2007/003711 _Cys86) pure article; And further obtaining the pure article of Interferon Alfacon-1 86 site cysteine mutant modified by polyethyleneglycol products (PEG-IIFNm1) of molecular weight homogeneous with embodiment 4, embodiment 5 disclosed raw materials and method, the product of separation and purification gained (also is disclosed mPEG (20KD)-MIFN among the PCT/CN2007/003711 behind the IIFNm for the mPEG-MAL of molecular weight 20kDa (modify Cys sulfydryl polyethyleneglycol modified dose) modifies _Cys86).

2, enzymolysis for the first time

Use molecular cut off to concentrate and change solution-treated the pure article of the above-mentioned PEG-IIFNm1 that obtains as the Millipore ultra-filtration centrifuge tube of 3000Da; The concentration of PEG-IIFNm1 is reached more than the 2mg/ml, and residing solution environmental converts 1% ammonium bicarbonate soln (pH8.0) into.Measure the concentration of the PEG-IIFNm1 behind the ultrafiltration and concentration with the Lowry method, and further to use 1% ammonium bicarbonate soln (pH8.0) to adjust its concentration be 2.00 ± 0.05mg/ml.

Get the above-mentioned sample that concentrates and adjust concentration of 1mg; Adding 8mol/L urea 0.5ml and 0.5mol/LDTT 0.1ml earlier handled 2 hours; Add 37 ℃ of enzyme digestion reactions of Trypsin25 μ g 18 hours of the commercial protein group classes and grades in school of SIGMA company then, add 100 μ l, 50% acetum enzymolysis reaction at last.The situation that contains the halfcystine fragment of theoretical connection PEG is as shown in table 1 below behind the Trypsin enzymolysis PEG-IIFNm1 under reducing condition.

The theoretical situation that contains the halfcystine fragment that connects PEG behind the Trypsin enzymolysis PEG-IIFNm1 under table 1 reducing condition

3, enzymolysis product separates for the first time

TSK-GEL G3000SW on the enzyme digestion reaction liquid _XL(performance liquid chromatographic column of 7.8mm * 30cm), 10mmol/LPB, 20mmol/LNaCl, the pH7.0 eluant solution, flow velocity 1.0ml/min detects wavelength 215nm, each appearance 100 μ l that go up.

Under the high performance liquid chromatography piece-rate system and separation condition identical with enzymatic fragment; Go up the pure article of IIFNm and PEG-IIFNm1 respectively; The result is as depicted in figs. 1 and 2 respectively; Confirm that retention time is respectively 9.564min and 7.032min, according to the present invention summary of the invention part described " retention time comparison method " confirm to connect polyalkylene glycol moiety enzymatic fragment go out the peak position, confirm between 7.032-9.564min, to go out the peak for connecting the enzymatic fragment peak of polyalkylene glycol moiety.

The high performance liquid chromatography of above-mentioned enzymolysis product has two peaks in separating between retention time 7.032-9.564min; As shown in Figure 3; Be the enzymatic fragment peak that connects polyalkylene glycol moiety, the collection retention time is 7.142min, and what peak area accounted for two peak area summations 85% goes out peak T1.

4, enzymolysis for the second time

Use molecular cut off to carry out concentration at the T1 peak of 3-5 high performance liquid chromatography separated and collected, and to change solution be 25mmol/L Tris-HCl, pH7.8 solution as the Millipore ultra-filtration centrifuge tube of 3000Da.

Get and above-mentionedly concentrate that to change solution-treated T1 appearance half the, carry out N terminal sequence mensuration with the Applied Biosystem PROCISE of company 491 type N terminal sequence analyzers.Sequencing result is FCTEL; What T1 peak correspondence was described is the 85-121 fragment that connects polyalkylene glycol moiety; But, be that Cys86 or Cys99 have connected polyalkylene glycol moiety on earth so be not sure of, or the both has connected polyalkylene glycol moiety owing in this fragment two Cys are arranged.

Second half adds Roche company 25 ℃ of enzyme digestion reactions of commercial order-checking level Chymotrypsin 5 μ g and adds 30 μ l, 50% acetum enzymolysis reaction after 18 hours.The situation that contains the halfcystine fragment of theoretical connection PEG is as shown in table 2 below after the Chymotrypsin enzymolysis PEG-IIFNm185-121 fragment under reducing condition.

The theoretical situation that contains the halfcystine fragment that connects PEG after the Chymotrypsin enzymolysis PEG-IIFNm185-121 fragment under table 2 reducing condition

The fragment position	Fragment sequence	Mass-to-charge ratio	Explanation
				97-111	EACVIQEVGVEETPL	1615.79	Cys99 belongs to fragment
86-89	CTEL	465.20	Cys86 belongs to fragment

5, the N terminal sequence of enzymolysis product separation for the second time and receipts appearance is measured

Enzyme digestion reaction liquid is gone up TSK-GEL G3000SW once more for the second time _XL(performance liquid chromatographic column of 7.8mm * 30cm), 10mmol/LPB, 20mmol/LNaCl; The pH7.0 eluant solution, flow velocity 1.0ml/min detects wavelength 215nm; Each appearance 100 μ l that go up are to go out peak C1 about 7.597min having only a retention time between the retention time 7.032min to 9.564min, and are as shown in Figure 4; For connecting the enzymatic fragment peak of polyalkylene glycol moiety, collect this peak.

The C1 peak is carried out the N terminal sequence with the Applied Biosystem PROCISE of company 491 type N terminal sequence analyzers to be measured.Sequencing result is CTE, explains that polyglycol has only connected Cys86 in the enzymatic fragment of the corresponding connection polyalkylene glycol moiety in T1 peak, does not connect Cys99, also both in the whole actual decorating site of PEG-IIFNm1, does not comprise Cys99.

Result in conjunction with twice high performance liquid chromatography separation confirms that Cys86 is the decorating site that has comparative advantage, and the PEG-IIFNm1 in this site of single modification shared ratio in the PEG-IIFNm1 of whole decorating sites is 85%.

Embodiment 2:PEG-IIFNm1 decorating site is analyzed (method two)

The acquisition of IIFNm and PEG-IIFNm1, for the first time enzymolysis, the step method measured of enzymolysis and N terminal sequence is with embodiment 1 for the second time, but twice enzymolysis product when separating all with WatersSymmetry 300C on the enzyme digestion reaction liquid ₁₈(4.6 * 150mm) reverse-phase chromatographic columns; Flow velocity 1.0ml/min detects wavelength 215nm, each appearance 100 μ l that go up; Gradient elution A liquid is the 0.1%TFA WS; B liquid is the acetonitrile solution of 0.1%TFA, rises to 90%B (10%A) linear gradient elution by 70%B (30%A) in 90 minutes when enzymolysis product separates for the first time, rises to 90%B (10%A) linear gradient elution by 30%B (70%A) in 15 minutes when enzymolysis product separates for the second time.Confirm to connect through summary of the invention part of the present invention described " electrophoresis dying method " polyalkylene glycol moiety enzymatic fragment go out the peak position.It is as shown in Figure 5 that enzymolysis product separates the chromatogram that obtains for the first time, and the chromatogram that enzymolysis product separation for the second time obtains is as shown in Figure 6.Separate the back at each RPLC and remove the organic solvent acetonitrile of receiving in the appearance with rotary evaporator, and then carry out that follow-up ultrafiltration and concentration is handled or the order-checking of N end is handled, the result is as shown in table 3 below.

The sequencing result and the analysis at each RPLC separated and collected peak behind twice enzymolysis of table 3PEG-IIFNm1

Can confirm equally that through said method Cys86 is the decorating site that has comparative advantage; The PEG-IIFNm1 in this site of single modification shared ratio in the PEG-IIFNm1 of whole decorating sites is 85%; And Cys99 is not actual decorating site; And can confirm further that Cys1 shared ratio in whole decorating sites is 6%, and Cys29 shared ratio in whole decorating sites is 6%, Cys139 shared ratio in whole decorating sites is 3%.

Embodiment 3: granulocyte colony stimulating factor modified by polyethyleneglycol product decorating site is analyzed

1, the acquisition of G-CSF and PEG-G-CSF

1) G-CSF expresses the engineering bacteria structure

Granulocyte colony stimulating factor (granulocyte colony stimulating factor; G-CSF) amino acid sequence is shown in SEQ ID No.2; Utilize commercial plasmid pET-23b and commercial e. coli bl21 (DE3); With the recombination engineering that well known to a person skilled in the art gene engineering method construction expression G-CSF (for example, the gene order of expression G-CSF is inserted between the Nde I and EcoR I restriction enzyme site of plasmid pET-23b).

2) G-CSF expresses the fermentation of engineering bacteria

With the G-CSF that makes up as stated above express engineering bacteria after being coated with dull and stereotyped activation, select single colony inoculation in the LB nutrient culture media that contains ampicillin 37 ℃, 220 rev/mins shaking table shake-flask culture to OD _600nmBe 0.6-0.8.Be inoculated in the 80L fermentation tank that 50L LB nutrient culture media is housed with 5% volume inoculum concentration then and carry out fermented and cultured, cultivation temperature is 37 ℃, regulates pH between 6.5-7.5 with ammoniacal liquor in the incubation, regulates speed of agitator control oxygen dissolving value between 3-5%.At OD _600nmReach 1.0 backs and add IPTG 10g in 1: 5000 ratio of mass volume ratio and continued inducing culture 4 hours, the inducing culture temperature is 35 ℃, and in this process in fermentation tank the suitable LB nutrient culture media of adding.The inducing culture time to after put 5000 rev/mins of jar room temperatures and collected thalline in centrifugal 20 minutes, the gained thalline with TE solution (50mmol/L Tris-HCl, 5mmol/L EDTA, pH8.0) wash centrifugal twice to remove the major impurity in the fermentation liquor.

3) purifying of G-CSF

Getting thalline 40g that above-mentioned processing obtains adds 600ml TE solution with 1: 15 mass volume ratio and puts and carry out ultrasonication on the ultrasonic disruption appearance; Condition is for to make a call to 5 seconds; Had a rest 5 seconds; Totally 60 minutes, 6000 rev/mins of broken suspension room temperatures of gained were abandoned supernatant in centrifugal 20 minutes, and deposition adds the TE solution washing with 1: 10 mass volume ratio must separate inclusion body centrifugal twice.

Gained 10g inclusion body added behind the 100ml 7mol/L guanidine hydrochloride under appropriate stirring condition degenerative treatments 2 hours with 1: 10 mass volume ratio; Treat that inclusion body dissolves 8000 rev/mins of the room temperatures in back fully and abandoned deposition in centrifugal 20 minutes; Supernatant carries out renaturation with the dilution refolding method to be handled, and renaturation solution consists of: 0.15mol/L boric acid-sodium borate, 3mmol/L oxidized form of glutathione; The 1mmol/L reduced glutathione, regulating pH is 9.5.Renaturation process carries out at 2-8 ℃ of low temperature, at first with renaturation solution with 4 times of supernatant dilutions, places after 8 hours and continued renaturation 6 hours for 5 times with the renaturation solution dilution again, and then with 5 times of renaturation solution dilutions, renaturation 6 hours is to reach final renaturation effect at last.4 ℃ 8000 rev/mins centrifugal renaturation solutions were 30 minutes after renaturation was accomplished, get supernatant by 1: 10 volume ratio to 25mmol/L Tris-HCl, pH8.0 solution carries out dialysis treatment, dialysis time is 12-24 hour, dislysate is changed 1-2 time in the centre.

Renaturation solution after the dialysis is gone up after centrifugal 30 minutes through 4 ℃ 8000 rev/mins and is used 25mmol/L Tris-HCl; The DEAE Sepharose FF chromatographic column of pH8.0 solution equilibria; With the 25mmol/LTris-HCl that contains 0.3mol/L NaCl, the pH8.0 eluant solution is also collected the peak that contains destination protein.Linear flow rate in the whole process should be controlled between the 50-200cm/h.

The peak that the collection of DEAE Sepharose FF wash-out contains destination protein is with 50mmol/L acetic acid-sodium acetate; PH4.5 solution is gone up the CM Sepharose FF chromatographic column with same solution equilibria with 1: 10 volume ratio dilution back; With the 25mmol/L acetic acid-sodium acetate that contains 0.1-0.15mol/L NaCl; The pH4.5 eluant solution is used the 25mmol/L acetic acid-sodium acetate that contains 0.3-0.5mol/L NaCl after removing the major impurity peak; PH4.5 eluant solution purpose is collected albumen, and the SDS-PAGE purity of the G-CSF that collects is more than 95%.Linear flow rate in the whole process should be controlled between the 50-200cm/h.

CM Sepharose FF wash-out is collected the peak that contains destination protein and is used 25mmol/L Tris-HCl; Go up once more behind 10 times of the pH8.5 solution dilutions and use 25mmol/L Tris-HCl; The DEAE Sepharose FF chromatographic column of the filler filling less (below the 10ml) of pH8.5 solution equilibria; With containing the 25mmol/LTris-HCl of 0.5mol/L NaCl, the pH8.5 eluant solution is so that G-CSF obtains PEG before modifying concentrates and change damping fluid and handle.

4) PEG modifies and the separating of modified outcome

Use the G-CSF pure article of the commercial molecular weight of Jiankai Science and Technology Co., Ltd., Beijing or Beijing Kaizheng Biotech Engineering Development Co., Ltd. as polyethyleneglycol modified dose of mPEG-SPA (amino polyethyleneglycol modified dose) the modification said method acquisition of 20kDa; Concrete modification reaction condition is: G-CSF concentration 5mg/ml (available 25mmol/L Tris-HCl; The concentration of the G-CSF that the last step of pH8.5 solution adjustment obtains is concentration so far); G-CSF and mPEG-SPA mass ratio 1: 5; Room temperature was carried out modification reaction 2 hours, left standstill and did not stir.

Modification reaction liquid is with 20mmol/L acetic acid-sodium acetate; Go up SPSepharose FF ion-exchange chromatography behind 20 times of the pH4.5 solution dilutions, the flushing back is earlier with the 20mmol/L acetic acid-sodium acetate that contains 70mmol/L NaCl, pH4.5 eluant solution impurity protein; Again with the 20mmol/L acetic acid-sodium acetate that contains 0.2mol/L NaCl; PH4.5 eluant solution purpose is collected albumen, at last with the 20mmol/L acetic acid-sodium acetate that contains 0.75mol/L NaCl, the not adorned G-CSF of pH4.5 eluant solution.Linear flow rate in the whole process should be controlled between the 30-100cm/h.

2, enzymolysis

Use molecular cut off to concentrate and change solution-treated the above-mentioned granulocyte colony stimulating factor modified by polyethyleneglycol product (PEG-G-CSF) that obtains as the Millipore ultra-filtration centrifuge tube of 3000Da; The concentration of PEG-G-CSF is reached more than the 2mg/ml, and residing solution environmental converts 1% ammonium bicarbonate soln (pH8.0) into.Measure the concentration of the PEG-G-CSF behind the ultrafiltration and concentration with the Lowry method, and further to use 1% ammonium bicarbonate soln (pH8.0) to adjust its concentration be 2.00 ± 0.05mg/ml.

Get the above-mentioned sample that concentrates and adjust concentration of 1mg; Adding 8mol/L urea 0.5ml and 0.5mol/LDTT 0.1ml earlier handled 2 hours; Add 37 ℃ of enzyme digestion reactions of Trypsin 25 μ g 24 hours of the commercial order-checking level of Promega company then, add 100 μ l, 50% acetum enzymolysis reaction at last.The situation that contains lysine blockage of theoretical connection PEG is as shown in table 4 below behind the Trypsin enzymolysis PEG-G-CSF under reducing condition.

The theoretical situation that connects the lysine blockage of PEG behind the Trypsin enzymolysis PEG-G-CSF under table 4 reducing condition

3, enzymolysis product separates the N terminal sequence mensuration of receiving appearance

Protein-Pak 200SW on the enzyme digestion reaction liquid (performance liquid chromatographic column of 8.0mm * 30cm), 10mmol/LPB, 20mmol/LNaCl, the pH7.0 eluant solution, flow velocity 1.0ml/min detects wavelength 215nm, each appearance 100 μ l that go up.

According to summary of the invention part of the present invention described " retention time comparison method "; Under same high performance liquid chromatography piece-rate system and separation condition, go up the pure article record of G-CSF and PEG-G-CSF retention time respectively; The gained chromatogram is respectively like Fig. 7 and shown in Figure 8; The retention time that shows both is for Wei not 9.432min and 6.954min, thereby the retention time of confirming to connect the enzymatic fragment of polyalkylene glycol moiety should be between 6.954-9.432min.

Between retention time 6.954-9.432min, have two peaks in the chromatographic resolution of above-mentioned enzymolysis product; Chromatogram is as shown in Figure 9; Be the enzymatic fragment peak that connects polyalkylene glycol moiety; Collecting retention time is about 7.560min, and what peak area accounted for two peak area summations 80% goes out peak T2, carries out N terminal sequence mensuration with the Applied Biosystem PROCISE of company 491 type N terminal sequence analyzers.

Sequencing result is KVQAD; What T2 peak correspondence was described is the 44-55 fragment that connects polyalkylene glycol moiety; Because have only an amino acid residue Lys44 that can connect polyalkylene glycol moiety in this fragment, so Lys44 is the decorating site that has comparative advantage among the PEG-G-CSF, proportion is 80%.

Embodiment 4: VEGF modified by polyethyleneglycol product decorating site is analyzed

1, the acquisition of VEGF and PEG-VEGF

1) the vegf expression engineering bacteria makes up

VEGF (vascular endothelial growth factor; VEGF) amino acid sequence is shown in SEQ ID No.3; Utilize commercial plasmid pET-23b and commercial e. coli bl21 (DE3); With the recombination engineering (for example, the gene order of VEGF expression is inserted between the Nde I and EcoR I restriction enzyme site of plasmid pET-23b) that well known to a person skilled in the art gene engineering method construction expression VEGF.

2) fermentation of vegf expression engineering bacteria

With the vegf expression engineering bacteria that makes up as stated above after being coated with dull and stereotyped activation, select single colony inoculation in the LB nutrient culture media that contains ampicillin 37 ℃, 220 rev/mins shaking table shake-flask culture to OD _600nmBe 0.6-0.8.Carry out fermented and cultured after being inoculated in the 200L fermentation tank that 140L LB nutrient culture media is housed with 6% volume inoculum concentration then, condition such as embodiment 3, but the inducing culture temperature is 36 ℃.The inducing culture time to after put 5000 rev/mins of jar room temperatures and collected thalline in centrifugal 20 minutes, the gained thalline with twice of above-mentioned TE solution washing to remove the major impurity in the fermentation liquor.

3) purifying of VEGF

The modification treatment of thalline ultrasonication and inclusion body is with embodiment 3.

Gained 10g inclusion body contains the 5mmol/L mercaptoethanol with 1: 15 mass volume ratio adding 150ml; The 50mol/L Tris-HCl of 8mol/L urea; Behind the pH8.0 solution under appropriate stirring condition degenerative treatments 4 hours; Treat that inclusion body dissolves 8000 rev/mins of the room temperatures in back fully and abandoned deposition in centrifugal 20 minutes, supernatant carries out renaturation and purification process with the on-column refolding method in room temperature.Concrete grammar is: the DEAE Sepharose FF chromatographic column of handling with the identical solution equilibria of equivariance solution on the supernatant, and 50mol/L Tris-HCl is used, 5mmol/L EDTA, 4M urea earlier in the flushing back; 3-5 column volume of pH8.0 eluant solution used 50mol/L Tris-HCl, 5mmol/LEDTA again; 2M urea, 3-5 column volume of pH8.0 eluant solution used 50mol/L Tris-HCl again; 5mmol/LEDTA, 1M urea, 3-5 column volume of pH8.0 eluant solution; Use 50mol/L Tris-HCl solution again, 3mmol/L oxidized form of glutathione, 1mmol/L reduced glutathione; 5mmol/L EDTA, 50mmol/L arginine, pH 8.0 an eluant solution 5-8 column volume; Using 50mmol/L Tris-HCl, with the 50mmol/L Tris-HCl that contains 0.3mol/L NaCl, the pH8.0 eluant solution is also collected the peak that contains destination protein behind 3-5 column volume of pH8.0 eluant solution.Linear flow rate in the whole process should be controlled between the 50-200cm/h.

DEAE Sepharose FF wash-out is collected the peak that contains destination protein and is mixed with 50% ammonium sulfate (mass and size concentration) by 2: 3 volume ratio; Using 1mol/L Tris-HCl to regulate pH is with the 50mmol/L Tris-HCl that contains 20% ammonium sulfate on 8.0 backs; Phenyl Sepharose 6FF (Low sub) chromatographic column of pH8.0 solution equilibria; Use 50mmol/L Tris-HCl, pH8.0 eluant solution purpose is collected albumen, and the SDS-PAGE purity of the VEGF that collects is more than 95%.Linear flow rate in the whole process should be controlled between the 50-200cm/h.

Phenyl Sepharose 6FF (Low sub) wash-out is collected on the peak that contains destination protein and is used 50mmol/LTris-HCl; The DEAE Sepharose FF chromatographic column of the filler filling less (below the 10ml) of pH8.0 solution equilibria; With the 50mmol/L Tris-HCl that contains 0.5mol/L NaCl, the pH8.0 eluant solution is so that VEGF obtains the concentration before PEG modifies.

4) PEG modifies and the separating of modified outcome

Use the VEGF pure article of the commercial molecular weight of Jiankai Science and Technology Co., Ltd., Beijing or Beijing Kaizheng Biotech Engineering Development Co., Ltd. as polyethyleneglycol modified dose of mPEG-NHS of Y type (amino polyethyleneglycol modified dose) modification said method acquisition of 40kDa; The modification reaction condition is VEGF concentration 4mg/ml (available 50mmol/L Tris-HCl; The concentration of the VEGF that the last step of pH8.0 solution adjustment obtains is concentration so far); VEGF and mPEG-NHS mass ratio 1: 6; Room temperature was carried out modification reaction 2 hours, 10 rev/mins of stirring rates.

Modification reaction liquid is with 20mmol/L acetic acid-sodium acetate; Go up the ToyopearlSP650M ion-exchange chromatography behind 20 times of the pH4.5 solution dilutions, the flushing back is earlier with the 20mmol/L acetic acid-sodium acetate that contains 50mmol/L NaCl, pH4.5 eluant solution impurity protein; Right in the 20mmol/L acetic acid-sodium acetate that contains 0.14mol/LNaCl; PH4.5 eluant solution purpose is collected albumen, at last with the 20mmol/L acetic acid-sodium acetate that contains 0.75mol/LNaCl, the not adorned VEGF of pH4.5 eluant solution.Linear flow rate in the whole process should be controlled between the 30-100cm/h.

2, enzymolysis

Use molecular cut off to concentrate and change solution-treated the above-mentioned VEGF modified by polyethyleneglycol product (PEG-VEGF) that obtains as the Millipore ultra-filtration centrifuge tube of 3000Da; The concentration of PEG-VEGF is reached more than the 2mg/ml, and residing solution environmental converts 1% ammonium bicarbonate soln (pH8.0) into.Measure the concentration of the PEG-VEGF behind the ultrafiltration and concentration with the Lowry method, and further to use 1% ammonium bicarbonate soln (pH8.0) to adjust its concentration be 2.00 ± 0.05mg/ml.

Get the above-mentioned sample that concentrates and adjust concentration of 1mg; Adding 8mol/L urea 0.5ml and 0.5mol/LDTT 0.1ml earlier handled 2 hours; Add 37 ℃ of enzyme digestion reactions of Trypsin50 μ g 24 hours of the commercial order-checking level of Luo Shi (Roche) company then, add 100 μ l, 50% acetum enzymolysis reaction at last.The situation that contains lysine blockage of theoretical connection PEG is as shown in table 5 below behind the Trypsin enzymolysis PEG-VEGF under reducing condition.

The theoretical situation that contains lysine blockage that connects PEG behind the Trypsin enzymolysis PEG-VEGF under table 5 reducing condition

3, enzymolysis product separates the N terminal sequence mensuration of receiving appearance

With Waters Symmetry 300C on the enzyme digestion reaction liquid ₁₈(4.6 * 150mm) reverse-phase chromatographic columns, flow velocity 1.0ml/min detect wavelength 215nm; Each appearance 100 μ l that go up; Gradient elution A liquid is the 0.1%TFA WS, and B liquid is the acetonitrile solution of 0.1%TFA, rises to 90%B (10%A) linear gradient elution by 70%B (30%A) in 90 minutes.The gained chromatogram is shown in figure 10, confirm to connect through summary of the invention part of the present invention described " electrophoresis dying method " polyalkylene glycol moiety each enzymatic fragment go out the peak position.Separate the back at RPLC and remove the organic solvent acetonitrile of receiving in the appearance with rotary evaporator, and then carry out follow-up N end order-checking and handle, the result is as shown in table 6 below.

The sequencing result and the analysis at the RPLC separated and collected peak behind the table 6PEG-VEGF enzymolysis

Can confirm that through said method Lys42 is the decorating site that has comparative advantage; The PEG-VEGF in this site of single modification shared ratio in the PEG-VEGF of whole decorating sites is 62%; And can confirm further that Lys22 shared ratio in whole decorating sites is 9%; Lys133 shared ratio in whole decorating sites is 8%, and Lys162 shared ratio in whole decorating sites is 9%, and Lys173 shared ratio in whole decorating sites is 12%.

Embodiment 5:PEG-IIFNm2 decorating site is analyzed (method three)

1, the acquisition of IIFNm and PEG-IIFNm2

The preparation method of IIFNm is with embodiment 1.The IIFNm that obtains is used 25mmol/L Tris-HCl; Go up behind 10 times of the pH8.5 solution dilutions and use 25mmol/L Tris-HCl; The DEAE Sepharose FF chromatographic column of the filler filling less (below the 10ml) of pH8.5 solution equilibria; With containing the 25mmol/L Tris-HCl of 0.5mol/L NaCl, the pH8.5 eluant solution is so that IIFNm obtains PEG before modifying concentrates and change damping fluid and handle.

Use polyethyleneglycol modified dose mPEG-SPA (amino polyethyleneglycol modified dose) the modification IIFNm of the commercial molecular weight of Jiankai Science and Technology Co., Ltd., Beijing or Beijing Kaizheng Biotech Engineering Development Co., Ltd. as 5kDa; Concrete modification reaction condition is: IIFNm concentration 5mg/ml (available 25mmol/L Tris-HCl; The concentration of the IIFNm that the last step of pH8.5 solution adjustment obtains is concentration so far); IIFNm and mPEG-SPA mass ratio 1: 8, room temperature carried out modification reaction 2 hours, left standstill and did not stir.

Modification reaction liquid is with 20mmol/L acetic acid-sodium acetate; Go up SPSepharose FF ion-exchange chromatography behind 20 times of the pH4.5 solution dilutions, the flushing back is earlier with the 20mmol/L acetic acid-sodium acetate that contains 0.1mol/L NaCl, pH4.5 eluant solution impurity protein; Again with the 20mmol/L acetic acid-sodium acetate that contains 0.25mol/L NaCl; PH4.5 eluant solution purpose is collected albumen, at last with the 20mmol/L acetic acid-sodium acetate that contains 0.75mol/L NaCl, the not adorned IIFNm of pH4.5 eluant solution.Linear flow rate in the whole process should be controlled between the 30-100cm/h.

2, enzymolysis for the first time

Use molecular cut off to concentrate and change solution-treated the above-mentioned PEG-IIFNm2 that obtains as the Millipore ultra-filtration centrifuge tube of 3000Da; The concentration of PEG-IIFNm2 is reached more than the 2mg/ml, and residing solution environmental converts 50mmol/L sodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH7.0) into.Measure the concentration of the PEG-IIFNm2 behind the ultrafiltration and concentration with the Lowry method, and further to use 50mmol/L sodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH7.0) to adjust its concentration be 2.00 ± 0.05mg/ml.

Get the above-mentioned sample that concentrates and adjust concentration of 1mg; Adding 8mol/L urea 0.5ml and 0.5mol/LDTT 0.1ml earlier handled 2 hours; Add the Glu-C 25 μ g37 ℃ enzyme digestion reactions 18 hours of the commercial order-checking level of Luo Shi (Roche) company then, add 100 μ l, 50% acetum enzymolysis reaction at last.Under reducing condition in the phosphate buffer behind the Glu-C enzymolysis PEG-IIFNm2 the theoretical situation that contains lysine blockage that connects PEG as shown in table 7 below.

The theoretical situation that contains lysine blockage that connects PEG behind the Glu-C enzymolysis PEG-IIFNm2 in the phosphate buffer under table 7 reducing condition

3, enzymolysis product separates for the first time

Waters Symmetry 300C on the enzyme digestion reaction liquid ₁₈(4.6 * 150mm) reverse-phase chromatographic columns, flow velocity 1.0ml/min detect wavelength 215nm; Each appearance 100 μ l that go up; Gradient elution A liquid is the 0.1%TFA WS, and B liquid is the acetonitrile solution of 0.1%TFA, rises to 80%B (20%A) linear gradient elution by 50%B (50%A) in 120 minutes.The order-checking of N end and each decorating site proportion grading result of the enzymatic fragment of connection polyglycol are as shown in table 8 below.

Table 8PEG-IIFNm2 enzymolysis for the first time separates sequencing result and the analysis that the peak is collected in the back with RPLC

4, enzymolysis and subsequent high performance liquid chromatography separate for the second time

It is that 3000 Millipore ultra-filtration centrifuge tube carries out concentration that the G3 peak of 3-5 high performance liquid chromatography separated and collected is used molecular cut off, and to change solution be 1% ammonium bicarbonate soln (pH8.0).The 37 ℃ of enzyme digestion reactions of Trypsin 5 μ g that in concentrating the G3 peak collection appearance of changing solution-treated, add the commercial protein group classes and grades in school of SIGMA company add 30 μ l, 50% acetum enzymolysis reaction after 18 hours.The situation that contains lysine blockage of theoretical connection PEG is as shown in table 9 below after the Trypsin enzymolysis PEG-IIFNm2134-142 fragment under reducing condition.

The theoretical situation that contains lysine blockage that connects PEG after the Trypsin enzymolysis PEG-IIFNm2134-142 fragment under table 9 reducing condition

The fragment position	Fragment sequence	Mass-to-charge ratio	Explanation
				134-135	KK	275.21	Lys134 belongs to fragment
134-142	KKYSPCAWE	1111.52	Lys135 belongs to fragment

Waters Symmetry 300C on the enzyme digestion reaction liquid for the second time ₁₈(4.6 * 150mm) reverse-phase chromatographic columns, flow velocity 1.0ml/min detect wavelength 215nm; Each appearance 100 μ l that go up; Gradient elution A liquid is the 0.1%TFA WS, and B liquid is the acetonitrile solution of 0.1%TFA, rises to 90%B (10%A) linear gradient elution by 30%B (70%A) in 15 minutes.The order-checking of N end and each decorating site proportion grading result of the enzymatic fragment of connection polyglycol are as shown in table 10 below.

Table 10Trypsin enzymolysis PEG-IIFNm2134-142 fragment is through sequencing result and the analysis of Trypsin enzymolysis with RPLC separates collection peak, back

5, enzymolysis and subsequent high performance liquid chromatography separate for the third time

It is that 3000 Millipore ultra-filtration centrifuge tube carries out concentration that the G6 peak of 3-5 high performance liquid chromatography separated and collected is used molecular cut off, and to change solution be 1% ammonium bicarbonate soln (pH8.0).The Trypsin 5 μ g37 ℃ enzyme digestion reactions that in concentrating the G6 peak collection appearance of changing solution-treated, add the commercial protein group classes and grades in school of SIGMA company add 30 μ l, 50% acetum enzymolysis reaction after 18 hours.The situation that contains lysine blockage of theoretical connection PEG is as shown in table 11 below after the Trypsin enzymolysis PEG-IIFNm2116-133 fragment under reducing condition.

The theoretical situation that contains lysine blockage that connects PEG after the Trypsin enzymolysis PEG-IIFNm2116-133 fragment under table 11 reducing condition

The fragment position	Fragment sequence	Mass-to-charge ratio	Explanation
				116-122	SILAVKK	758.51	Lys121 belongs to fragment
116-126	SILAVKKYFQR	1352.80	Lys122 belongs to fragment

Waters Symmetry 300C on the enzyme digestion reaction liquid for the third time ₁₈(4.6 * 150mm) reverse-phase chromatographic columns, flow velocity 1.0ml/min detect wavelength 215nm; Each appearance 100 μ l that go up; Gradient elution A liquid is the 0.1%TFA WS, and B liquid is the acetonitrile solution of 0.1%TFA, rises to 90%B (10%A) linear gradient elution by 30%B (70%A) in 15 minutes.The order-checking of N end and each decorating site proportion grading result of the enzymatic fragment of connection polyglycol are as shown in table 12 below.

Table 12Trypsin enzymolysis PEG-IIFNm2116-133 fragment is through sequencing result and the analysis of Trypsin enzymolysis with RPLC separates collection peak, back

Through as above the experiment and interpretation of result show that Lys31 is a dominant decorating site among the PEG-IIFNm2, ratio is 33%, decorating site that other is less important and ratio are: Lys165,15%; Lys122,12.4%; Lys84,11%; Lys50,8%; Lys71,7%; Lys135,5.6%; Lys121,4.6%; Lys134,3.4%.

The structure of the polyethyleneglycol modified dose of mPEG-MAL that uses in the foregoing description is following:

The structure of mPEG-SPA is following:

The structure of Y type mPEG-NHS is following:

Claims (10)

1. the decorating site analytical approach of polyethyleneglycol modified protein comprises the steps: at least

1) enzymolysis: primary enzymolysis at least, under enzymolysis fully and the condition that does not have non-specific enzymolysis, each possible decorating site is distributed in the different theoretical enzymatic fragments;

2) high performance liquid chromatography separates: at least high performance liquid chromatography separates behind each enzymolysis; Be distributed in the different chromatographic peaks with the relatively actual enzymatic fragment that does not connect polyalkylene glycol moiety of the enzymatic fragment that finally makes actual connection polyalkylene glycol moiety; And finally make at least include only dominant actual decorating site residue on the content on the sequence and do not comprise maybe but non-actual decorating site residue and connect include only actual decorating site residue less important on the same content on each sequence of enzymatic fragment comparison of polyalkylene glycol moiety and do not comprise maybe but non-actual decorating site residue and connect the enzymatic fragment of polyalkylene glycol moiety be distributed in the different chromatographic peaks;

3) confirm the decorating site ratio: to include only dominant actual decorating site residue on the content on the sequence and do not comprise maybe but include only actual decorating site residue less important on the same content on non-actual decorating site residue and the enzymatic fragment that connects polyalkylene glycol moiety and each sequence and do not comprise maybe but non-actual decorating site residue and the enzymatic fragment that connects polyalkylene glycol moiety 2) chromatographic resolution in finally corresponding chromatographic resolution peak carry out peak area and add and handle; Not the peak area of the chromatographic peak that obtains of homogeneous chromatographic resolution need by add again after converting with the peak area of a chromatographic resolution with; And confirm that the maximum chromatographic resolution peak of peak area wherein accounts for that peak area adds and ratio, to confirm dominant actual decorating site proportion in whole decorating sites;

4) confirm the decorating site position: collect include only dominant actual decorating site residue on the content on the sequence and do not comprise maybe but non-actual decorating site residue and the sample that connects the final corresponding chromatographic resolution peak of the enzymatic fragment institute of polyalkylene glycol moiety carry out the N terminal sequence measures, with the theoretical enzymolysis sequence definite position of dominant actual decorating site in protein sequence of comparing.

2. analytical approach as claimed in claim 1 is characterized in that enzyme that each time enzymolysis in the enzymolysis step is selected is one or more the combination among Trypsin, Chymotrypsin, Glu-C, the Asp-N.

3. analytical approach as claimed in claim 1 is characterized in that each high performance liquid chromatography separates to select a kind of in RPLC post or the molecular exclusion performance liquid chromatographic column.

4. analytical approach as claimed in claim 3 is characterized in that described RPLC post selection Waters Symmetry 300C ₁₈Chromatographic column.

5. analytical approach as claimed in claim 3 is characterized in that described molecular exclusion performance liquid chromatographic column selection TSK-GEL G3000SW _XLChromatographic column.

6. analytical approach as claimed in claim 1 is characterized in that described protein is Interferon Alfacon-1 86 site cysteine mutants, and it has the amino acid sequence shown in the SEQ ID No.1.

7. analytical approach as claimed in claim 6 is characterized in that comprising the steps:

1) Trypsin primary enzymolysis;

2) high performance liquid chromatography of primary enzymolysis product separates; So that connecting the enzymatic fragment of polyalkylene glycol moiety, each reality of enzymatic fragment comparison of each actual connection polyalkylene glycol moiety is not distributed in the different chromatographic peaks; And the enzymatic fragment of each actual connection polyalkylene glycol moiety in same site also is distributed in the different chromatographic peaks; Collect the sample at each peak that peak area has comparative advantage in the corresponding chromatographic peak of the enzymatic fragment of the actual connection in same site polyalkylene glycol moiety; And through the peak area that calculates this peak account for adding of the corresponding chromatographic peak peak areas of the enzymatic fragment of whole each actual connection polyalkylene glycol moieties in same site and ratio, thereby confirm actual decorating site proportion in whole decorating sites of having comparative advantage;

3) to 2) in the peak sample collected carry out the Chymotrypsin secondary enzymolysis;

4) high performance liquid chromatography of secondary enzymolysis product separates, and collects the enzymatic fragment peak that connects polyalkylene glycol moiety;

5) the enzymatic fragment peak of twice high performance liquid chromatography separated and collected is carried out the N end sequence and measure, through confirming the position of actual decorating site in Interferon Alfacon-1 86 site cysteine mutant sequences that have comparative advantage with theoretical enzymolysis sequence contrast.

8. method as claimed in claim 7 is characterized in that step 2) and/or step 4) described in high performance liquid chromatography separate to adopt Waters Symmetry 300C ₁₈The RPLC post.

9. method as claimed in claim 7 is characterized in that step 2) and/or step 4) described in high performance liquid chromatography separate to adopt TSK-GEL G3000SW _XLThe molecular exclusion performance liquid chromatographic column.

10. like the described analytical approach of one of claim 1-6, following steps are further required:

1) high performance liquid chromatography separates: at least high performance liquid chromatography separates behind each enzymolysis, with finally make include only actual decorating site residue less important on the same content on each sequence and do not comprise maybe but non-actual decorating site residue and the enzymatic fragment that connects polyalkylene glycol moiety be distributed in the different chromatographic peaks;

2) confirm the decorating site ratio: to include only actual decorating site residue less important on the same content on each sequence and do not comprise maybe but the non-actual decorating site residue and the peak area of chromatographic peak that connects the enzymatic fragment place of polyalkylene glycol moiety add and handle; Calculate include only actual decorating site residue less important on the same content on each sequence and do not comprise maybe but the non-actual decorating site residue and the peak area of chromatographic peak that connects the enzymatic fragment place of polyalkylene glycol moiety account for include only actual decorating site residue less important on the same content on whole each sequences and do not comprise maybe but the non-actual decorating site residue and the peak area of chromatographic peak that connects the enzymatic fragment place of polyalkylene glycol moiety add and ratio, to confirm each actual decorating site proportion in whole decorating sites;

3) confirm the decorating site position: collect include only actual decorating site residue less important on the same content on each sequence and do not comprise maybe but the non-actual decorating site residue and the sample at chromatographic resolution peak that connects the enzymatic fragment place of polyalkylene glycol moiety carry out the N terminal sequence measures, compare with theoretical enzymolysis sequence and confirm each actual decorating site position in protein sequence.

Images